Growth Factor-beta Research Articles

Nicotinic acetylcholine receptor activation mediates nicotine‐induced enhancement of experimental periodontitis

Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. This study was designed to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Ligature-induced periodontitis was applied in periodontitis-susceptible Fischer 344 rats. The animals were either given daily intraperitoneal injections of the nicotinic acetylcholine receptor antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous injections in the neck skin of nicotine (0.8 mg/kg), or treated with the same amount of saline intraperitoneally and nicotine subcutaneously, or treated with mecamylamine and saline. Control animals received intraperitoneal and subcutaneous injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 wk. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 microg/kg, intraperitoneally) to induce a robust immune and stress response. Compared with saline/saline-treated control animals, saline/nicotine-treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor-alpha, transforming growth factor-1beta and interleukin-10. Mecamylamine pretreatment of nicotine-treated rats abrogated the increased periodontal bone loss and the LPS-induced decrease in tumour necrosis factor-alpha, but had no significant effects on the levels of transforming growth factor-1beta and interleukin-10, or the stress hormone corticosterone. The results indicate that nicotine enhances the susceptibility to periodontitis via nicotinic acetylcholine receptors, which may act by suppressing protective immune responses through the cholinergic anti-inflammatory pathway.

Inhibition of TGFβ1 Makes Nonimmunogenic Tumor Cells Effective for Therapeutic Vaccination

We have previously demonstrated that transplanted cells from the SW1 clone of the K1735 melanoma and the Ag104 sarcoma grow progressively in syngeneic C3H mice even after transfection to engage CD137, a procedure that increases the immunogenicity of many other tumors. We now show that SW1 and Ag104 cells produce high levels of transforming growth factor (TGF) beta1, and that they can induce an antitumor response if they are transfected with a nonreplicating lentivirus vector to "silence" the TGFbeta1 gene via short hairpin RNA. Importantly, vaccination with SW1 or Ag104 cells, which do not make TGFbeta1, is therapeutically efficacious against small wild type tumors, including SW1 micrometastases in the lung. An analogous approach may be applicable to human tumors that produce TGFbeta1 or other immunosuppressive molecules to improve the efficacy of tumor cell-based therapeutic vaccines.

Expression of macrophage inhibitory cytokine‐1 in prostate cancer bone metastases induces osteoclast activation and weight loss

Macrophage inhibitory cytokine-1 (MIC-1) belongs to the bone morphogenic protein/transforming growth factor-beta (BMP/TGF-beta) superfamily. Serum MIC-1 concentrations are elevated in patients with advanced prostate cancer. The effects of MIC-1 on prostate cancer bone metastases are unknown. In vitro effects of MIC-1 on osteoblast differentiation and activity were analyzed with alkaline phosphatase and mineralization assays; osteoclast numbers were counted microscopically. MIC-1 effects on TLR9 expression were studied with Western blotting. Human Du-145 prostate cancer cells were stably transfected with a cDNA encoding for mature MIC-1 or with an empty vector. The in vivo growth characteristics of the characterized cells were studied with the intra-tibial model of bone metastasis. Tumor associated bone changes were viewed with X-rays, histology, and histomorphometry. Bone formation was assayed by measuring serum PINP. MIC-1 induced osteoblast differentiation and activity and osteoclast formation in vitro. These effects were independent of TLR9 expression, which was promoted by MIC-1. Both MIC-1 and control tumors induced mixed sclerotic/lytic bone lesions, but MIC-1 increased the osteolytic component of tumors. Osteoclast formation at the tumor-bone interface was significantly higher in the MIC-1 tumors, whereas bone formation was significantly higher in the control mice. At sacrifice, the mice bearing MIC-1 tumors were significantly lighter with significantly smaller tumors. MIC-1 up-regulates TLR9 expression in various cells. MIC-1 stimulates both osteoblast and osteoclast differentiation in vitro, independently of TLR9. MIC-1 over-expressing prostate cancer cells that grow in bone induce osteoclast formation and cachexia.

Nicotinic acetylcholine receptor activation mediates nicotine‐induced enhancement of experimental periodontitis

Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. Our objective was to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Ligature-induced periodontitis was applied in periodontitis-susceptible Fischer 344 rats. The animals were given daily intraperiotonal (i.p.) injections of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous (s.c.) injections in the neck skin with nicotine (0.8 mg/kg), or treated with the same amount of saline i.p. and nicotine s.c., or with mecamylamine and saline. Control rats received i.p. and s.c. injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 weeks. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 microg/kg, i.p.) to induce a robust immune and stress response. Compared with saline/saline-treated control rats, saline/nicotine-treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor alpha (TNF-alpha), transforming growth factor 1beta (TGF-1beta) and interleukin-10 (IL-10). Mecamylamine pretreatment of nicotine-treated rats abrogated the increased periodontal bone loss and the LPS-induced TNF-alpha decrease, but had no significant effects on the levels of TGF-1beta and IL-10, or the stress hormone corticosterone. The results indicate that nicotine enhances susceptibility to periodontitis via nAChRs, which may act via suppressing protective immune responses through the cholinergic anti-inflammatory pathway.

Attenuation of diabetic nephropathy by tocotrienol: Involvement of NFkB signaling pathway

Aim Diabetic nephropathy is a serious complication for patients with diabetes mellitus. Approximately 30–40% of patients with type I and 15% with type II diabetes mellitus develop end stage renal disease. The study was designed to evaluate the impact of tocotrienol on renal function and reno-inflammatory cascade in streptozotocin-induced diabetes. Main methods Streptozotocin (STZ)-induced diabetic rats were treated with tocotrienol (25, 50 and 100 mg/kg), α-tocopherol (100 mg/kg) or with vehicle form 5th to 8th weeks. After 8 weeks, urine albumin excretion, urine output, serum creatinine, blood urea nitrogen, creatinine and urea clearance were measured. Cytoplasmic and nuclear fractions of kidney was prepared for the quantification of oxidative–nitrosative stress (lipid peroxidation, superoxide dismutase, catalase, non protein thiols, total nitric oxide), tumor necrosis factor-alpha (TNF-α), tissue growth factor-1beta (TGF-β1), p65 subunit of NFκβ and caspase-3. Key findings After 8 weeks of STZ injection, the rats produced significant alteration in renal function, increased oxidative–nitrosative stress, TNF-α, TGF-β1, caspase-3 activity in cytoplasmic lysate and active p65 subunit of NFκβ in nuclear lysate of kidney of diabetic rats. Interestingly, co-administration of tocotrienol significantly and dose-dependently prevented biochemical and molecular changes associated with diabetes. Tocotrienol (100 mg/kg) was demonstrated to be more effective than α-tocopherol (100 mg/kg). Moreover, diabetic rats treated with insulin-tocotrienol combination produced more pronounced effect on molecular parameters as compared to their respective groups. Significance Taken together, the data reveal that tocotrienol modulates the release of profibrotic cytokines, oxidative stress, ongoing chronic inflammation and apoptosis and thus exerts a marked renoprotective effect.

Telmisartan prevents aneurysm progression in the rat by inhibiting proteolysis, apoptosis and inflammation

We investigated the effects of treatment with the angiotensin II type 1 receptor antagonist, telmisartan, on abdominal aortic aneurysm formation in normotensive rats. Abdominal aortic aneurysm was induced by perfusion of an isolated aortic segment with elastase. Treatment with telmisattan (0.5 mg/kg per day) or hydralazine (15 mg/kg per day) was started after surgery and continued for 14 days. Sham-operated animals and vehicle-treated animals after aneurysm induction served as controls. Aortic diameter was measured using ultrasound before aneurysm induction and on days 7 and 14 after aneurysm induction. On day 14, aortic diameter was increased two-fold in the vehicle-treated group compared to sham-operated animals (2.02 +/- 0.12 vs. 0.87 +/- 0.02 mm, P < 0.005, n = 8). Telmisartan treatment significantly reduced aneurysmal size (1.65 +/- 0.06 vs. 2.02 +/- 0.12 mm in vehicle, P < 0.05, n = 8), whereas treatment with hydralazine had no effect. Matrix metallopeptidase 3, cathepsin D, nuclear factor kappa B, tumour necrosis factor alpha, transforming growth factor-1 beta, as well as caspase 3, p53 and Fas ligand proteins, were significantly downregulated in aortic tissue under telmisartan compared to vehicle treatment. Serum monocyte chemoattractant protein 1 levels were also significantly decreased. Telmisartan and hydralazine reduced blood pressure to a similar extent within the observation period. The angiotensin II type 1 receptor antagonist, telmisartan, prevents abdominal aortic aneurysm progression independently of blood pressure reduction by inhibiting proteolysis, apoptosis and inflammation in aortic tissue.

Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory effect of interleukin-1β

To investigate the effects of resveratrol on interleukin-1beta (IL-1beta) induced catabolism in bone marrow mesenchymal stem cell (MSC) derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS). The chondrogenesis of alginate-encapsulated MSCs was evaluated by toluidine blue staining, RT-PCR, and immunostaing. MSC-derived chondrocyte morphology cultured on CGS was evaluated by a scanning electron microscope (SEM) and a laser confocal microscope (LCM). When these cells on CGS were pre-stimulated with IL-1beta or co-treated with IL-1beta and resveratrol in the absence and presence of the specific beta1-integrin blocking antibody, collagen type II, aggrecan, matrix metalloproteinase-13 (MMP-13) expression, and the translocation of nuclear factor kappaB (NF-kappaB) were analyzed by Western blot analysis. Transforming growth factor beta 3 (TGF-beta3) combined with insulin-like growth factor I (IGF-I) induced the cartilage-specific collagen type II, aggrecan expression and extracellular matrix (ECM) accumulation at the end of a 3-week culture. CGS supported those differentiated chondrocytes'attachment, proliferation, migration, and ECM formation. When those cells cultured on CGS were stimulated with IL-beta1 alone, collagen type II and aggrecan expression was inhibited. However, MMP-13 expression increased. Resveratrol reversed the catabolic effects by reducing the translocation of NF-kappaB. A specific beta1-integrin blocking antibody abrogated the effects of resveratrol on IL-1beta stimulated MSC-derived chondrocytes. These results indicated that resveratrol acts as a NF-kappaB inhibitor to protect MSC-derived chondrocytes on the CGS from the IL-1beta catabolism and these effects are mediated by beta1-integrin.

Cloning of the eukaryotic expression vector with nerve growth factor in rats and its effects on proliferation and differentiation of mesencephal neural stem cells of fetal rats

The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor (NGF) beta subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF beta subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGF. The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF beta was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and facilitate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF beta subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.

Cytokine Array After Cyclosporine Treatment in Rats

Objectives Long-term treatment with cyclosporine (CsA) results in chronic nephrotoxicity, which is known to be mediated by several cytokines including transforming growth factor-betal. Cytokines are known to play an important role in innate immunity, apoptosis, angiogenesis, cell growth, and differentiation. They are known to be involved in most disease processes, including cancer, cardiac disease, and nephrotoxicity. To evaluate changes of cytokines in a rat model of CsA-induced chronic nephrotoxicity, we performed a cytokine array. Methods Experiments were performed on two groups of rats; normal control group and CsA-treated group. Cytokine array in rat serum was performed using Cytokine Antibody Array I kit from RayBiotech. Results Serum creatinine, urine creatinine, and creatinine clearance increased in the CsA-treated group. Among the several cytokines, the expressions of the lipopolysaccharide-induced CXC chemokine (LIX), monocyte chemoattractant protein 1 (MCP-1), nerve growth factor (β-NGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the CsA-treated group were increased above that of cytokines in the control group. The density of the LIX in controls was 0.62, and in the CsA-treated group was 1.24. The density of the MCP-1 in controls was 0.68, and in CsA-treated, 1.43. The density of the β-NGF in controls was 0.62, and that in CsA-treated, 1.24. The density of the TIMP-1 in controls 1.13, and in CsA-treated, 1.40. Conclusions Our data suggested that among several cytokines elevated levels of the LIX, MCP-1, β-NGF, and TIMP-1 are the contributing factors to CsA-induced nephropathy.

The antioxidant effect exerted by TGF-1β-stimulated hyaluronan production reduced NF-kB activation and apoptosis in human fibroblasts exposed to FeSo4 plus ascorbate

Previous studies suggest that Transforming growth factor-1beta (TGF-1beta) administration in human fibroblasts exposed to oxidative stress is able to modulate hyaluronan synthases (HASs). HAS modulation in turn increases high molecular weight (Hyaluronan) HA concentration. Nuclear factor kB (NF-kB) is a response transcription factor involved in inflammation and acts by enabling the expression of certain detrimental molecules. Caspases are specific proteases responsible for regulating and programming cell death. HA at medium molecular weight together with chondroitin-4-sulphate proved to be effective on NF-kB and caspases. We investigated whether the protective effect afforded by the high molecular weight HA produced by TGF-1beta treatment has any effect on NF-kB and apoptosis activation in fibroblast cultures exposed to oxidative stress. Generation of free radicals gives rise to cell death, increases lipid peroxidation, activates NF-kB, reduces its cytoplasmic inhibitor IkBalpha, augments caspase-3 and caspase-7 gene expression and their relative protein activity, and depletes catalase (CAT) and glutathione peroxidase (GPx). Treatment of fibroblasts with TGF-1beta 12 h before inducing oxidative stress greatly increased HA levels, ameliorated cell survival, inhibited lipid peroxidation, blunted NF-kB translocation, normalized IkBalpha protein, reduced caspase gene expression and protein levels, and restored the endogenous antioxidants CAT and GPx. Since it was previously reported that antioxidants can work as inhibitors of NF-kB and apoptosis induction we can hypothesize that endogenous HA, by inhibiting lipid peroxidation, may block a step whereby free radical activity converges in the signal transduction pathway leading to NF-kB and caspase activation.

Breast Cyst Fluid Plasmin Activity and Its Effect on TGF-β2 Activation

There are two types of breast cyst and women with apocrine breast cyst may have a higher risk of developing breast cancer than cyst lined by flattened epithelium. Transforming growth factor-beta's growth inhibitory effect on epithelial cells suggests a potential protective role in breast cancer. The aim of this study was to investigate the presence of plasmin in both breast cyst groups and the possible role of plasmin on transforming growth factor beta activation. Presence of high plasmin level may indicate its importance on activation process, but some other proteases may also involve in this activation mechanism.

Abstract 2187: The Balance Of Pro-inflammatory And Ant-inflammatory Circulating Cytokine mRNA Gene Expression In CHF Patients With And Without Cachexia Compared To Healthy Control Subjects.

Background: Chronic heart failure (CHF) is a state of chronic inflammation. High serum levels of TNF alpha and the presence of cardiac cachexia are known to predict poor survival in CHF. Abnormal balance of pro- and anti-inflammatory circulating cytokine mRNA gene expression is thought to contribute to cachexia in humans, but has never been studied in CHF patients. Methods and Results: We studied 7 patients with cardiac cachexia (age 70±4 years, LVEF 28±2%, peak VO2 12.7±1.6 mL/kg/min, all &gt;6% weight loss in previous 6–24 months), 19 noncachectic CHF patients (age 68±2 years, LVEF 31±2%, peak VO2 14.7±1.1 mL/kg/min) and 17 control subjects of similar age and gender (peak VO2 25.1±1.7 mL/kg/min, LVEF 71±2%). The mRNA gene expression of circulating mononuclear blood cells for pro-inflammatory mediators that have been implicated in the wasting process of ageing (TNF, STAT-1, STAT-3, SOCS-1 and SOCS-3) as well as anti-inflammatory cytokines (IL-10, tumor growth factor (TGF) beta were investigated by real time polymerase chain reaction. Values for the house keeping gene and genes of interest were calculated from double determinations within 40 cycles. We found higher TNF expression in cachectic compared to noncachectic CHF patients (log mean −0.5±0.17 vs. −1.2±0.12, p=0.002, ANOVA-p=0.0071) and control subjects (log mean −1.2±0.1, p=0.006). In contrast, there was no change in IL-10 gene expression in cachectic patients compared to noncachectic patients and controls (log mean −2.6±0.2 vs. −3.0±0.09 vs. −2.9±0.1, all p&gt;0.13). Consequently TNF/IL-10 expression ratio was highest in cachectic patients (308±138 vs. 132±55 in noncachectic CHF patients vs. 101±37 in controls, p&lt;0.004). There were no differences in gene expression of STAT-1, STAT-3, SOCS-1, SOCS-3 between cachectic and noncachectic individuals (all p&gt;0.17). Similarly, no differences were found for gene expression of TGF beta (p&gt;0.15). Conclusion: The balance between the pro-inflammatory cytokine TNF and the anti-inflammatory cytokine IL-10 on the level of mRNA expression of circulating mononuclear cells is significantly disturbed. Proinflammatory cytokine imbalance in cardiac cachexia may be important for the pathophysiology of body wasting and may represent the activated immune reflex.

Albumin-stimulated TGFβ-1 in renal tubular cells is associated with activation of MAP kinase

Proteinuric renal diseases are often associated with progressive tubulointerstitial fibrosis that usually defines the degree and rate of progression of renal failure. Glomerular filtration of excess albumin, the dominant protein in proteinuria, into proximal tubule could provide the stimulus to induce certain fibrogenic cytokines from proximal tubular cells (PTC), which may account for fibrosis in the interstitium. To explore this hypothesis we tested the effect of bovine albumin in PTC in culture on the expression and secretion of transforming growth factor (TGF) beta-1, a prominent fibrogenic cytokine. TGFbeta-1 expressed by cultured PTC was measured by enzyme-linked immunosorbent assay (ELISA) and indirectly by reverse-transcription polymerase chain reaction (RT-PCR). Relative messenger ribonucleic acid (mRNA) levels were measured in ethidium bromides stained gels, by comparison to transcripts for 18s ribosomal RNA. Activated mitogen-activated protein (MAP) kinase was estimated by Western blot with phosphotyrosine-specific antibody. Following incubation of PTC with albumin determination of TGF beta-1 mRNA in PTC and TGF beta-protein in culture medium both indicated a time- and dose-dependent increase. MAP kinase (p44/42) was activated within 5 min of exposure to albumin. Inhibition of the MAP kinase cascade by PD98059 attenuated the effect of albumin on TGF beta-1 expression. These observations suggest that overexpression of TGF beta-1 by PTC in response to albumin is regulated through a MAP kinase signaling pathway. This mechanism may play a role in the development of interstitial fibrosis in proteinuric states.

Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy.

Effects of silicon dioxide on expression of alpha-smooth muscle actin in human lung fibroblasts

To investigate the effects of SiO(2) on the expression of alpha-smooth muscle actin (alpha-SMA) in human lung fibroblasts in vitro and vivo. The experimental group comprised 32 rats while 32 rats were included in the control. In vivo, the expression of alpha-SMA in lung tissues of rats exposed to SiO(2), the supernate of RAW264.7 cells, SiO(2) and the growth factor beta(1) (TGF-beta(1)) were investigated, respectively. (1) alpha-SMA positive myofibroblasts appeared in the lung tissues of the 28th day groups exposed to SiO(2). (2) The expression of alpha-SMA in HLF-02 cells was unregulated by TGF-beta(1) and supernate of RAW264.7 cells exposed to SiO(2). (3) The expression of alpha-SMA in HLF-02 cells was not induced by SiO(2). Myofibroblasts related to silicosis, and the appearance of myofibroblasts (in vitro) are independent on direct stimulation by SiO(2), but related to the mediator (TGF-beta(1)) secreted by SiO(2) stimulated macrophages.

Design and screening of antisense oligodeoxynucleotides against PAI-1 mRNA in endothelial cells in vitro1

To design and screen antisense oligodeoxynucleotides (ASODNs), which inhibit type-1 plasminogen activator inhibitor (PAI-1) expression in human umbilical vein endothelial cells (HUVEC) in vitro. Twenty seven ASODNs against different sites of PAI-1 mRNA were designed and transfected to HUVEC by lipofectin in vitro. The effects of ASODNs on PAI-1 antigen, PAI-1 activity and PAI-1 mRNA expression were detected by ELISA, amidolytical assay and RT-PCR, respectively. Transforming growth factor beta1 (TGF-beta1)-treated HUVEC increased the expression of PAI-1 compared with the normal HUVEC. Five among twenty seven designed ASODNs were effective in inhibiting the increase in PAI-1 antigen and PAI-1 activity in a dose-dependent manner after 48-h transfection. In particular, ASODN 14 (AO14) exhibited the best inhibitory effect. The control sequences of AO14, including sense, scramble, and mismatch sequences, did not significantly inhibit PAI-1 activity. It was revealed that the inhibitory efficacy of AO14 was in a sequence-specific manner. RT-PCR showed that ASODN 1, 7, 8, 14, and 15 decreased PAI-1 mRNA expression induced by TGF-beta1 and AO14 showed the best inhibitory effect. ASODN 1, 7, 8, 14, and 15, among twenty seven designed ASODNs against PAI-1 mRNA, significantly decreased PAI-1 antigen and PAI-1 activity induced by TGF-beta1 in a dose-dependent manner in HUVEC in vitro. AO14 showed the best inhibitory effect on PAI-1 expression in a sequence-specific manner. The results of RT-PCR indicated that inhibitory effects of ASODNs on PAI-1 biosynthesis occurred at the mRNA level. Four among five effective target sites of ASODNs located at the translation initiation site or within the translation area of PAI-1 mRNA, suggesting that these sites may be promising sites for the design of effective ASODNs.

Effects of ethanol and transforming growth factor beta (TGF beta) on neuronal proliferation and nCAM expression.

BACKGROUND Developmental events targeted by ethanol are cell proliferation, neuronal migration, and neurite outgrowth; the latter processes being mediated by neural cell adhesion molecule (nCAM). TGFbeta1 affects all three of these events. Therefore, the effects of ethanol on transforming growth factor (TGF) beta1 mediated activities in neocortical neurons in vitro were examined. METHODS Primary cultures of cortical neurons were obtained from 16-day-old fetuses and were treated with TGFbeta1 (0 or 10 ng/ml) and ethanol (0 or 400 mg/dl) for 48 hr. The effects of these substances on cell numbers, [ H]thymidine incorporation, and the expression of nCAM were determined. RESULTS Both cell growth (the change in cell numbers over time) and cell proliferation were inhibited by TGFbeta1 and ethanol. The action of these two anti-mitogenic factors was additive. In contrast, TGFbeta1 also promoted the expression of three isoforms of nCAM. Likewise, ethanol also up-regulated nCAM expression. On the other hand, ethanol blocked TGFbeta1-mediated nCAM expression, particularly of the 120 and 180 kDa isoforms. CONCLUSIONS TGFbeta ligands inhibit neuronal proliferation and stimulate the expression of cell adhesion proteins that promote the movement of postmitotic neurons and process outgrowth. Ethanol alters these phenomena as well. Thus, in neurons, as in astrocytes, TGFbeta1 and ethanol may interact.

Die diagnostische und prädiktive Bedeutung von Kit (CD117)

KIT (CD117) is a 145-KD transmembrane glycoprotein that is the product of the kit-gene. As a member of the subclass III family of receptor tyrosine kinases, KIT is closely related to the receptors for platelet derived growth factor alpha and beta (PDGF-A and B), macrophage colony stimulating factor (M-CSF), and FLT3 ligand. The ligand for KIT, stem cell factor (SCF), also known as steel factor or mast cell growth factor promotes the dimerisation and autophosphorylation of KIT receptors. The phosphorylated tyrosine residues provide binding sites for signal molecules that contain SH2 domains. KIT mediated signal transduction is critical for the normal development and survival of haematopoietic progenitor cells, mast cells, interstitial cells of Cajal (intestinal pacemaker cells), melanocytes and germ cells. Upon differentiation, KIT expression is lost with the exception of mast cells, melanocytes and interstitial cells of Cajal. The detection of CD117 expression is of paramount diagnostic relevance in gastrointestinal stromal tumors (GIST). About 95% of all GISTs are immunohistochemically CD117 positive. The vast majority of all other sarcoma, carcinoma and also lymphoma are CD117 negative. Therefore, CD117 expression has a high sensitivity and specificity for the diagnosis of GIST. Moreover, activating mutations of KIT tyrosine kinase play a crucial pathogenetic role in GIST 80 to 85% of all GIST's contain activating mutations, primarily in Exon's 11 and 9 of the kit gene. Since the resulting mutant isoformes are sensitive to inhibition by imatinib (glivec), a specific tyrosine kinase inhibitor, the detection of a specific mutation has also a high predictive value. Besides GIST mastocytoses and seminomas are the neoplasias that most commonly express CD117. In contrast to GIST however, these two neoplasias contain mutations in different exons and are only partly imatinib sensitive. Moreover, CD117 expression is by no means entirely specific for these entities. It can also be detected in adenoid cystic carcinomas, thymic carcinomas and melanomas. Very rarely (< 5%) it can also be observed in other carcinomas and sarcomas. However, in the great majority of these cases with a CD117 protein expression there is no corresponding gene mutation of kit. Importantly, the lack of an activating mutation of KIT tyrosine kinase is good evidence that imatinib will not be effective. In other words, detection of sole CD117 protein expression is no solid basis for targeted therapy. The molecular pathological detection of CD117 expression in combination with the corresponding mutational status in patients with GIST (and other tumors) paradigmatically highlights the importance of modern molecular diagnostics in the era of targeted therapy.

Heterophil cytokine mRNA profiles from genetically distinct lines of chickens with differential heterophil-mediated innate immune responses

Previously we demonstrated that increased in-vitro heterophil function translates to increased in-vivo resistance to Salmonella enteritidis infections in broilers (line A > B). Heterophils produce cytokines and modulate acute protection against Salmonella in neonatal poultry. We hypothesized that heterophils from S. enteritidis-resistant chickens produce an up-regulated pro-inflammatory cytokine/chemokine response compared with S. enteritidis-susceptible chickens. In this study, heterophils were isolated 1, 14, and 28 days post-hatch, treated with RPMI or phagocytic agonists, and the cytokine/chemokine mRNA expression assessed using quantitative real-time reverse transcriptase-polymerase chain reaction. At all time-points, heterophils from S. enteritidis-resistant chickens (line A) had higher levels of pro-inflammatory cytokine/chemokine mRNA expression upon stimulation compared with heterophils from S. enteritidis-susceptible chickens (line B). Furthermore, heterophils from line A chickens had decreased mRNA expression of transforming growth factor-beta4, an anti-inflammatory cytokine, compared with line B. These data indicate a relationship between cytokine/chemokine mRNA expression by heterophils and determining overall immune competence. Therefore, heterophil functional efficiency, accompanied by evaluating cytokine/chemokines produced by heterophils, may be useful biomarkers for breeders to consider when developing new immunocompetent lines.

Gains of COL1A1-PDGFB genomic copies occur in fibrosarcomatous transformation of dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans is a superficial low-grade sarcoma that rarely evolves into a high-grade fibrosarcoma. Dermatofibrosarcoma protuberans is genetically characterized by the unbalanced chromosomal t(17;22)(q21;q13), usually in the form of a supernumerary ring chromosome. The product of this chromosomal translocation is the chimeric gene COL1A1-PDGFB (collagen type I alpha I-platelet-derived growth factor beta), which is amplified at low levels in the ring chromosome. The aims of this study were to evaluate (1) whether genomic gains of this fusion gene occur during the clonal evolution of dermatofibrosarcoma protuberans into fibrosarcomatous dermatofibrosarcoma protuberans and (2) whether there is a difference between the number of genomic copies of COL1A1-PDGFB between classic dermatofibrosarcoma protuberans and dermatofibrosarcoma protuberans areas associated with fibrosarcomatous dermatofibrosarcoma protuberans. Eleven cases of fibrosarcomatous dermatofibrosarcoma protuberans with both dermatofibrosarcoma protuberans and fibrosarcomatous areas and 10 cases of classic dermatofibrosarcoma protuberans were studied. Genomic copies of COL1A1-PDGFB were evaluated by fluorescence in situ hybridization using a custom designed probe for the PDGFB locus on 4 μm thick paraffin-embedded tissue sections. Genomic gains of the COL1A1-PDGFB gene were observed in six (of 10) fibrosarcomatous dermatofibrosarcoma protuberans in the fibrosarcomatous areas when compared to the dermatofibrosarcoma protuberans areas of the same tumor (2–7 gene copies (median PDGFB copy gain, 2.8) versus 1–3 gene copies (median PDGFB copy gain, 1.7), respectively, P=0.004). Four fibrosarcomatous dermatofibrosarcoma protuberans did not show genomic gains of COL1A1-PDGFB fusion gene between the two areas. Essentially no difference in the copy number of COL1A1-PDGFB fusion gene was observed between dermatofibrosarcoma protuberans areas of classic dermatofibrosarcoma protuberans and dermatofibrosarcoma protuberans areas of fibrosarcomatous dermatofibrosarcoma protuberans (median PDGFB copy gain of 1.8 versus 1.7, respectively, P=0.36). Genomic gains of COL1A1-PDGFB fusion gene is possibly an oncogenic mechanism that is identified in the clonal evolution of a subset of dermatofibrosarcoma protuberans that evolves into fibrosarcomatous dermatofibrosarcoma protuberans. Since this finding was not observed in all cases of fibrosarcomatous dermatofibrosarcoma protuberans, other oncogenic mechanisms may be operating in this form of tumor progression. Copy number of COL1A1-PDGFB fusion gene in the classic dermatofibrosarcoma protuberans areas does not seem to be a major predisposing mechanism for fibrosarcomatous transformation.